The angiotensin IV analog Nle-Tyr-Leu-psi-(CH2-NH2)3-4-His-Pro-Phe (norleual) can act as a hepatocyte growth factor/c-Met inhibitor.

The angiotensin (Ang) IV analog norleual [Nle-Tyr-Leu-psi-(CH2-NH2)(3-4)-His-Pro-Phe] exhibits structural homology with the hinge (linker) region of hepatocyte growth factor (HGF) and is hypothesized to act as a hinge region mimic. Norleual competitively inhibited the binding of HGF to its receptor c-Met in mouse liver membranes, with an IC(50) value of 3 pM. Predictably, norleual was able to inhibit HGF-dependent signaling, proliferation, migration, and invasion in multiple cell types at concentrations in the picomolar range. Ex vivo studies demonstrated that norleual exhibited potent antiangiogenic activity, an attribute that would be predicted for a HGF/c-Met antagonist. Furthermore, norleual suppressed pulmonary colonization by B16-F10 murine melanoma cells, which are characterized by an overactive HGF/c-Met system. Together, these data suggest that AngIV analogs exert at least some of their biological activity through interference with the HGF/c-Met system and may have utility as therapeutic agents in disorders that are dependent on an intact HGF/c-Met system. Finally, the ability of norleual to induce marked biological responses in human embryonic kidney cells, which do not express insulin-responsive aminopeptidase (IRAP), coupled with the observed effects of norleual on the HGF/c-Met system, casts doubt on the physiological significance of AngIV-dependent inhibition of IRAP. [Corrected]

AT4 receptor activation increases intracellular calcium influx and induces a non-N-methyl-D-aspartate dependent form of long-term potentiation.

The angiotensin 4 receptor (AT4) subtype is heavily distributed in the dentate gyrus and CA1-CA3 subfields of the hippocampus. Neuronal pathways connecting these subfields are believed to be activated during learning and memory processing. ur laboratory previously demonstrated that application of the AT4 agonist, Norleucine1-angiotensin IV, enhanced baseline synaptic transmission and long-term potentiation, whereas perfusion with the AT4 antagonist, Norleucine1-Leu3-psi(CH2-NH2)3-4-angiotensin IV disrupted long-term potentiation stabilization in area CA1. The objective of the present study was to identify the mechanism(s) responsible for Norleucine1-angiotensin IV-induced increase in hippocampal long-term potentiation. Hippocampal slices perfused with Norleucine1-angiotensin IV for 20 min revealed a notable increase in baseline responses in a non-reversible manner and were blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt. Infusions of Norleucine1-angiotensin IV prior to, but not after theta burst stimulation, significantly enhanced long-term potentiation compared with control slices. Further, N-methyl-D-aspartate receptor-independent long-term potentiation could be induced by tetanization during the perfusion of Norleucine1-angiotensin IV in the presence of the N-methyl-D-aspartate antagonist, D,L-2-amino-5-phosphonovaleric acid. Blockade of select voltage dependent calcium channels significantly reduced Norleucine1-angiotensin IV-induced increase in baseline responses and subsequent long-term potentiation suggesting that AT4 receptor activation increases intracellular calcium levels via altering voltage dependent calcium channels and triggers an N-methyl-D-aspartate-independent form of long-term potentiation. In support of this notion the application of Nle1-angiotensin IV to cultured rat hippocampal neurons resulted in increased intracellular calcium derived exclusively from extracellular sources. Consistent with these observations Nle1-angiotensin IV was capable of augmenting the uptake of 45Ca2+ into rat hippocampal slices. Taken together, these data indicate that increased calcium influx through postsynaptic calcium channels contribute to Norleucine1-angiotensin IV-induced enhancement of long-term potentiation.

A role for the angiotensin AT4 receptor subtype in overcoming scopolamine-induced spatial memory deficits.

There is increasing interest in the role of the brain angiotensin AT4 receptor subtype in cognitive processing. This receptor subtype is activated by angiotensin IV (AngIV), is heavily distributed in the mammalian hippocampus, neocortex, and cerebellum, and has been linked with a learning and memory function. The present investigation utilized intracerebroventricular (i.c.v.)-infused scopolamine hydrobromide (scop), a muscarinic receptor antagonist, to disrupt acquisition of the circular water maze task of spatial memory. All animals received 2 days of training trials (five trials/day) using a visible platform in an effort to preclude subsequent confounding by scopolamine-induced sensory and/or motor impairments. In the first experiment, i.c.v.-infused scopolamine (70 nmol) was followed by 0, 10, 100, or 1000 pmol i.c.v. doses of Nle(1)-AngIV in separate groups of rats. Results indicated that each dose of Nle(1)-AngIV improved the poor acquisition of this task induced by scopolamine treatment. However, the 100- and 1000-pmol doses were most effective with respect to latency and distance to find the submerged pedestal. A second experiment demonstrated that treatment with a specific AT4 receptor antagonist, Nle(1), Leual(3)-AngIV (1000 pmol), blocked the ability of Nle(1)-AngIV (100 pmol) to improve the performance of scopolamine-compromised rats. These results support the notion that hippocampal AT4 receptors are involved in spatial memory processing, and that activation of these binding sites can overcome the disruption of spatial memory accompanying treatment with a muscarinic receptor antagonist.

Conditions that affect sleep alter the expression of molecules associated with synaptic plasticity.

Many theories propose that sleep serves a purpose in synaptic plasticity. We tested the hypothesis, therefore, that manipulation of sleep would affect the expression of molecules known to be involved in synaptic plasticity. mRNA expression of four molecules [brain-derived neurotrophic factor (BDNF), activity-regulated cytoskeleton-associated protein (Arc), matrix metalloproteinase-9 (MMP-9), and tissue plasminogen activator (tPA)] was determined after 8 h of sleep deprivation and after 6 h of a mild increase in ambient temperature, a condition that enhances sleep in rats. After sleep deprivation, BDNF, Arc, and tPA mRNAs in the cerebral cortex increased while MMP-9 mRNA levels decreased. Conversely, after enhanced ambient temperature, BDNF, Arc, and tPA mRNAs decreased while MMP-9 mRNA increased. In the hippocampus, sleep deprivation did not significantly affect BDNF and tPA expression, although Arc mRNA increased and MMP-9 mRNA decreased. Brain temperature enhancement decreased Arc mRNA levels in the hippocampus but did not affect BDNF, MMP-9, or tPA in this area. Results are consistent with the notion that sleep plays a role in synaptic plasticity.

Angiotensin IV enhances LTP in rat dentate gyrus in vivo.

Angiotensins have been shown to play a significant role in a variety of physiological functions including learning and memory processes. Relatively recent evidence supports the increasing importance of angiotensin IV (Ang IV), in many of these functions previously associated only with Ang II, including learning and memory. An interesting hypothesis generated by these results has been that Ang II is a precursor for the production of a more active peptide fragment, Ang IV. Since Ang II impairs learning and memory, when administered directly or released into the hippocampal dentate gyrus, and inhibits long term potentiation (LTP) in medial perforant path-dentate granule cell synapses, as well; it remained to be seen what effects Ang IV had on LTP in these same synapses. Results of this study show clearly that Ang IV significantly enhances LTP, and the enhancement is both dose and time dependent. The following solutions of Ang IV were administered over a five min period, at the end of baseline and before the first tetanus was applied: 2.39, 4.78, and 9.56 nM. An inverted U-type dose related effect was observed. A complex time related effect was observed with a maximum at 5 min, a return to normal LTP at 30 min and a minimum below normal at 90 min, and a return to normal LTP at 120 min. The effects of the 4.78 nM solution were determined at the following intervals between administration and the first tetanus: 5, 15, 30, 60, 90, and 120 min. The enhancement of LTP can be prevented by pretreatment with Divalinal, an Ang IV antagonist, without any effect on normal LTP. Two solutions of Divalinal were used; 5 nM and 5 microM, and the 5 microM was more effective and completely blocked the enhancement of normal LTP. Results were also obtained with 4.78 nM Nle1-Ang IV (Norleucine), an Ang IV agonist. Norleucine was less effective than Ang IV in the enhancement of normal LTP and displayed a similar time course of activity. Both Ang IV and Norleucine produced a significant suppression of normal LTP at 90 min; that remains to be explained. However, the inhibition by Ang IV was dose dependent and was blocked by Divalinal. The fact that the Ang IV enhancement of normal LTP was blocked by losartan, an Ang II AT1 receptor antagonist, is puzzling since Divalinal had no effect on the inhibition of LTP by Ang II.

A role for the angiotensin IV/AT4 system in mediating natriuresis in the rat.

Angiotensin II (AngII) or Angiotensin IV (AngIV) was infused into the renal artery of anesthetized rats while renal cortical blood flow was measured via laser Doppler flowmetry. The infusion of AngII produced a significant elevation in mean arterial pressure (MAP) with an accompanying decrease in cortical blood flow, glomerular filtration rate (GFR), urine volume, and urine sodium excretion. The infusion of AngIV induced significant increases in renal cortical blood flow and urine sodium excretion, without altering MAP, GFR, and urine volume. Pretreatment infusion with a specific AT1 receptor antagonist, DuP 753, blocked or attenuated the subsequent AngII effects, while pretreatment infusion with the specific AT4 receptor antagonist, Divalinal-AngIV, blocked the AngIV effects. These results support distinct and opposite roles for AngII and AngIV, i.e. AngII acts as an anti-natriuretic agent, while AngIV acts as a natriuretic agent.

Analysis of response class hierarchies with attention-maintained problem behaviors.

We replicated a method for clarifying inconclusive functional analysis outcomes via an extinction analysis of separate topographies of problem behavior with 2 participants. Results suggested that both mild and severe problem behaviors belonged to the same response class. An analysis of response latency was consistent with a response class hierarchy hypothesis, indicating that mild problem behavior nearly always occurred prior to severe topographies of problem behavior.

The effects of angiotensin IV analogs on long-term potentiation within the CA1 region of the hippocampus in vitro.

Within the brain-renin angiotensin system, it is generally assumed that angiotensin peptide fragments shorter than angiotensins II and III, including angiotensin IV (AngIV), are inactive. This belief has been challenged by the recent discovery that AngIV, and AngIV-like analogs, bind with high affinity and specificity to a putative angiotensin binding site termed AT4. In the brain these sites include the hippocampus, cerebellum, and cerebral cortex, and influence associative and spatial learning tasks. The present study investigated the effects of two AngIV analogs, Nle1-AngIV (an AT4 receptor agonist) and Nle1-Leual3-AngIV (an AT4 receptor antagonist), on long-term potentiation (LTP). Field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 stratum radiatum following stimulation of the Schaffer collateral pathway. Activation of AT4 receptors by Nle1-AngIV enhanced synaptic transmission during low-frequency test pulses (0.1 Hz), and increased the level of tetanus-induced LTP by 63% over that measured under control conditions. Paired stimulation before and during infusion of Nle1-AngIV indicated no change in paired-pulse facilitation (PPF) as a result of AT4 receptor activation suggesting that the underlying mechanism(s) responsible for Nle1-AngIV-induced increase in synaptic transmission and LTP is likely a postsynaptic event. Further, applications of Nle1-Leual3-AngIV prior to, but not 15 or 30 min after, tetanization prevented stabilization of LTP. These results extend previous findings from behavioral data in that AT4 receptor agonists and antagonists are capable of activating, and inhibiting, learning and memory pathways in the hippocampus, and suggest that the AT4 receptor subtype is involved in synaptic plasticity.

Radiolabeling of Angiotensin peptides.

The use of radioiodinated angiotensins has contributed greatly to our knowledge of the renin-angiotensin system (RAS). This chapter provides brief descriptions of the application of radioiodinated angiotensins and other radioiodinated compounds to study the RAS and the issues that must be considered when using radioiodinated angiotensins. In addition, this chapter provides a detailed description of a method for the preparation and purification of both iodine(125) ((125)I) and iodine(127) ((127)I)-labeled angiotensin-related ligands.

Evaluating the effects of functional communication training in the presence and absence of establishing operations.

We conducted functional analyses of aberrant behavior with 4 children with developmental disabilities. We then implemented functional communication training (FCT) by using different mands across two contexts, one in which the establishing operation (EO) that was relevant to the function of aberrant behavior was present and one in which the EO that was relevant to the function of aberrant behavior was absent. The mand used in the EO-present context served the same function as aberrant behavior, and the mand used in the EO-absent context served a different function than the one identified via the functional analysis. In addition, a free-play (control) condition was conducted for all children. Increases in relevant manding were observed in the EO-present context for 3 of the 4 participants. Decreases in aberrant behavior were achieved by the end of the treatment analysis for all 4 participants. Irrelevant mands were rarely observed in the EO-absent context for 3 of the 4 participants. Evaluating the effectiveness of FCT across different contexts allowed a further analysis of manding when the establishing operations were present or absent. The contributions of this study to the understanding of functional equivalence are also discussed.

A novel angiotensin analog with subnanomolar affinity for angiotensin-converting enzyme.

This study demonstrates that a novel angiotensin I analog, angiotensinogen 3-11(Lys(11)), possesses a high affinity for angiotensin-converting enzyme (ACE), which is substantially greater than the endogenous substrates. This assessment is based on data derived from a variety of techniques. First, the binding characteristics of (125)I-angiotensinogen 3-11(Lys(11)) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that (125)I-angiotensinogen 3-11(Lys(11)) bound a single high-affinity site in the presence of EDTA exhibiting a K(d) of 0.15 +/- 0.02 nM with a B(max) = 4295 +/- 535 fmol/mg of protein. Competition studies revealed the following rank order of binding affinity: (125)I-angiotensinogen 3-11(Lys(11)) >> bradykinin >> angiotensin I. Next, SDS-polyacrylamide gel electrophoresis analysis revealed that chemically cross-linked (125)I-angiotensinogen 3-11(Lys(11)) specifically bound a protein of M(r) 173,000 that had the same molecular weight as ACE. Utilizing in vitro autoradiography, the binding distributions of (125)I-angiotensinogen 3-11(Lys(11)) and the ACE inhibitor, (125)I-351A, were also compared. These experiments demonstrated that the binding distributions of (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A binding. These results demonstrated that the binding site for (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A copurified. These experiments indicate that the novel angiotensin I analog, (125)I-angiotensinogen 3-11(Lys(11)) binds to ACE and suggest that there are critical binding sites outside the catalytic domains of ACE that determine binding specificity and affinity.

Involvement of nicotinic receptors in alcohol self-administration.

BACKGROUND: Alcohol and nicotine, in the form of tobacco, are commonly co-abused. Nicotinic receptors also have been implicated in alcohol action. We designed the present study to examine the possible involvement of nicotinic receptors in alcohol self-administration. METHODS AND RESULTS: Pretreatment with lower doses (0.1-0.4 mg/kg) of nicotine, administered acutely or chronically, did not affect alcohol consumption, whereas a higher dose (0.8 mg/kg) initially suppressed alcohol consumption but stimulated alcohol consumption on repeated treatment. We observed the same pattern of nicotine effects on alcohol self-administration using an operant procedure. A dose of 0.8 mg/kg of nicotine initially suppressed operant responding for alcohol. Such suppression of alcohol self-administration was more pronounced during the first 20 min of the 60 min operant session. Responding for alcohol in the nicotine treated group, however, was significantly increased above the saline treated group by the 5th day of treatment. Mecamylamine, a noncompetitive nicotinic receptor antagonist, reduced alcohol consumption, whereas dihydro-beta-erythroidine (DHbetaE), a competitive nicotinic receptor antagonist, did not modify alcohol consumption. CONCLUSIONS: The stimulation of alcohol intake induced by nicotine treatment and the suppression of alcohol intake induced by mecamylamine provide evidence for the involvement of nicotinic receptors in alcohol consumption and/or self-administration. The failure of DHbetaE to reduce alcohol consumption, however, suggests that ethanol-nicotine interaction is mediated by other nicotinic receptor subtypes rather than alpha4beta2 receptor subtype, or that mecamylamine acts through a nonnicotinic mechanism.

Characterization and function of the bovine kidney epithelial angiotensin receptor subtype 4 using angiotensin IV and divalinal angiotensin IV as receptor ligands.

125I-Angiotensin (Ang) IV and (125)I-divalinal Ang IV [AT receptor subtype 4 (AT(4))] receptor agonist and putative antagonist, respectively] were used to characterize the AT(4) receptor in Mardin-Darby bovine kidney epithelial cells (MDBK cell line). Both (125)I-Ang IV and (125)I-divalinal Ang IV bound to a single high-affinity site (K(D) = 1.37 and 1.01 nM, respectively) and to a comparable density of binding sites (B(max) = 1335 and 1407 fmol/mg protein, respectively). Competition of either radiolabeled ligand with several Ang related peptides demonstrated similar displacement affinities in the following affinity order: Ang IV = divalinal Ang IV > Ang III > Ang II > losartan = PD 123177. Guanosine-5'-O-(3-thio)triphosphate or sulfhydryl reducing agents did not affect the binding of either radiolabeled ligand. Brief exposure of MDBK cells to Ang IV or divalinal Ang IV (0.1 nM to 1 microM) caused a concentration-dependent rise in intracellular calcium concentration levels with a reduced calcium response observed with Ang IV at micromolar concentrations. These results indicate that Ang IV and divalinal Ang IV bind with high affinity to the same receptor and that the MDBK AT(4) receptor is not coupled to a classic G protein, nor are sulfhydryl bonds important in regulation of receptor affinity. The MDBK AT(4) receptor appears to be pharmacologically similar to that described in nonrenal tissues. Functional studies suggest that AT(4) receptor activation can increase intracellular calcium concentration levels in MDBK cells and that divalinal Ang IV possesses agonist activity with respect to this particular intracellular signaling system.

Angiotensin IV has mixed effects on left ventricle systolic function and speeds relaxation.

OBJECTIVE: A novel angiotensin receptor has been described and named AT4. Ligands for this receptor include the angiotensin II (Ang II) metabolite Ang II (3-8), known as angiotensin IV (Ang IV). There is 10-fold more AT4 receptor than AT1 receptor in rabbit myocardium. The AT4 receptor has a high affinity for Ang IV (Ki in rabbit myocardium < 2 x 10(-9)) and similar ligands, but very low affinity for Ang II (Ki in rabbit myocardium > 10(-6)). Although several functions have been attributed to the novel Ang IV peptide/AT4 receptor system, the effect of this system on left ventricular (LV) function has not been studied. We hypothesized (1) that Ang IV would affect LV function and (2) that any effects would be opposite to those of Ang II. METHODS: Using the buffer-perfused (30 degrees C) isolated rabbit heart, we studied the effect of the AT4 agonist Nle1-Ang IV on LV systolic function, quantified using both Frank-Starling and end-systolic pressure-volume relationships, and relaxation. We also studied the effect of the AT1/AT2 agonist, Sar1-Ang II on LV function. Finally, because the profile of effect of Nle1-Ang IV was similar to the reported effect of nitric oxide (NO), we also studied the effect of Nle1-Ang IV in the presence of the NO synthase inhibitor NG-monomethyl-L-arginine. RESULTS: Nle1-Ang IV reduced LV pressure-generating capability at any volume but increased the sensitivity of pressure development to volume change. Nle1-Ang IV reduced LV ejection capability. Sar1-Ang II had the opposite effect-increasing both pressure generation and ejection capability. Finally, both Sar1-Ang II and Nle1-Ang IV speeded LV relaxation. Inhibition of NO synthase did not alter the effect of Nle1-Ang IV on LV systolic function or relaxation. CONCLUSIONS: AT4 receptor agonism has mixed effects on LV systolic function, depressing pressure-generation and ejection capabilities, but enhancing the sensitivity of pressure development to volume change. It also speeds relaxation. The effect of Ang IV on systolic function is generally opposite to the effect of Ang II, whereas the Ang IV influence on relaxation is similar to the effect of Ang II.

Structure-binding studies of the adrenal AT4 receptor: analysis of position two- and three-modified angiotensin IV analogs.

Amino acid substitutions in positions two and three of angiotensin IV (VYIHPF) were carried out to determine which structural features of the side-chains were important for achieving high-affinity binding to bovine adrenal receptors. These studies demonstrated that an activated aromatic ring in the second position side-chain resulted in the highest-affinity binding. Position three required a hydrophobic amino acid to achieve high-affinity binding. Both aliphatic and aromatic side-chains were sufficient to yield high-affinity binding.

Contributions of the brain angiotensin IV-AT4 receptor subtype system to spatial learning.

The development of navigational strategies to solve spatial problems appears to be dependent on an intact hippocampal formation. The circular water maze task requires the animal to use extramaze spatial cues to locate a pedestal positioned just below the surface of the water. Presently, we investigated the role of a recently discovered brain angiotensin receptor subtype (AT4) in the acquisition of this spatial learning task. The AT4 receptor subtype is activated by angiotensin IV (AngIV) rather than angiotensins II or III, as documented for the AT1 and AT2 receptor subtypes, and is heavily distributed in the CA1-CA3 fields of the hippocampus. Chronic intracerebroventricular infusion of a newly synthesized AT4 agonist (Norleucine1-AngIV) via osmotic pump facilitated the rate of acquisition to solve this task, whereas treatment with an AT4 receptor antagonist (Divalinal) significantly interfered with the acquisition of successful search strategies. Animals prepared with bilateral knife cuts of the perforant path, a major afferent hippocampal fiber bundle originating in the entorhinal cortex, displayed deficits in solving this task. This performance deficit could be reversed with acute intracerebroventricular infusion of a second AT4 receptor agonist (Norleucinal). These results suggest that the brain AngIV-AT4 system plays a role in the formation of spatial search strategies and memories. Further, application of an AT4 receptor agonist compensated for spatial memory deficits in performance accompanying perforant path knife cuts. Possible mechanisms underlying this compensatory effect are discussed.

Structural analysis of angiotensin IV receptor (AT4) from selected bovine tissues.

The angiotensin IV receptor (AT4) receptor is widely distributed in both species and tissues. This broad distribution appears to be reflected in an equally diverse repertoire of physiological actions that are mediated through AT4 receptors. This breadth of location and function of AT4 receptors encourages speculation that multiple AT4 isoforms might exist. In this study, we compared the structural properties of bovine AT4 receptors from adrenals, kidney, heart, thymus, bladder, aorta, and hippocampus. These comparisons were made using polyacrylamide gel electrophoresis or HPLC analysis of AT4 receptors that had been covalently radiolabeled with the AT4-specific photoprobe 125I-benzoyl phenylalamine-angiotensin IV. Except for the hippocampal AT4 receptor, the binding subunit in all tissues had a molecular mass of approximately 165 kDa and associated with additional subunits via disulfide linkages. The hippocampal receptor was significantly smaller (150 kDa) and did not appear to possess other disulfide-linked subunits. The receptor was highly glycosylated in all tissues examined. Peptide mapping following cleavage of 125I-labeled receptor with endopeptidase C or cyanogen bromide resulted in complex cleavage patterns. Together these mapping studies demonstrated the uniqueness of the hippocampal receptor and further suggested that other AT4 isoforms may exist and be variably distributed among bovine tissues. In agreement with the peptide mapping studies, differences in the binding pattern of several AngIV analogs were observed among the various tissues.

An analysis of choice making in the assessment of young children with severe behavior problems.

We examined how positive and negative reinforcement influenced time allocation, occurrence of problem behavior, and completion of parent instructions during a concurrent choice assessment with 2 preschool-aged children who displayed severe problem behavior in their homes. The children were given a series of concurrent choice options that varied availability of parent attention, access to preferred toys, and presentation of parent instructions. The results showed that both children consistently allocated their time to choice areas that included parent attention when no instructions were presented. When parent attention choice areas included the presentation of instructions, the children displayed differential patterns of behavior that appeared to be influenced by the presence or absence of preferred toys. The results extended previous applications of reinforcer assessment procedures by analyzing the relative influence of both positive and negative reinforcement within a concurrent-operants paradigm.

Purification of two equine pepsinogens by use of high-performance liquid chromatography.

OBJECTIVES: To purify and characterize pepsinogens in equine gastric mucosa. SAMPLE POPULATION: Stomachs collected from 2 healthy horses at necropsy. PROCEDURE: After collection, stomachs were placed immediately in ice before storage at -48 C. After slow thawing, the mucosa was scraped off while the tissue was immersed in 0.1M potassium phosphate (pH 7.4) at 4 C, then was homogenized. The filtered extract was subjected to anion-exchange chromatography. Fractions that were found to contain pepsin or pepsinogen were further chromatographed. Individual fractions were tested for pepsinogen or pepsin content by monitoring proteolytic activity at pH 2 and 3, respectively. Fractions from all columns were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to confirm molecular weight of pepsinogens and pepsin. RESULTS: Two pepsinogens and at least 1 pepsin were purified from equine gastric mucosa. CONCLUSIONS: On the basis of molecular mass, equine gastric mucosa contains 2 pepsinogens. CLINICAL RELEVANCE: Results of this study will enable future development of an ELISA or radioimmunoassay for use in the diagnosis of equine gastric ulceration.

Sex differences in discriminative stimulus and diuretic effects of the kappa opioid agonist U69,593 in the rat.

Female and male rats were trained to discriminate the kappa opioid agonist (5alpha,7alpha,8beta)-(-)-N-methyl-[7-(1-pyrrolidinyl) -1-oxaspiro(4,5)dec-8-yl]benzeneacetamide (U69,593, 0.13 mg/kg SC) from vehicle using a FR-10 schedule of food reinforcement. Female rats took significantly longer than males to acquire the discrimination (66.9 vs. 44.1 sessions, respectively), and the ED50 for U69,593 discrimination was significantly higher in females than in males (0.074 vs. 0.025 mg/kg). The time course of U69,593 discrimination also differed between the sexes: peak and offset occurred earlier in females than in males. The ED50 for bremazocine substitution was significantly higher in females than in males (0.0039 vs. 0.0006 mg/kg), whereas ethylketazocine substituted for U69,593 in all males and five of seven females, with no sex difference in substitution ED50. Morphine and BW373U86 did not substitute for U69,593 in a majority of rats of either sex. U69,593 also produced significantly less urine output/dose in females compared to males (e.g., 5.92 vs. 14.83 ml urine/kg body weight after 1.0 mg/kg U69,593), but was equipotent between the sexes in producing hot-plate antinociception. There was no sex difference in response rate-decreasing effect of any opioid agonist tested, and no sex difference in brain/blood ratio of [3H]U69,593 measured in a separate group of rats, suggesting that sex differences observed in some effects of U69,593 probably are not due to sex differences in U69,593 pharmacokinetics. When retested at the end of the study, U69,593 and bremazocine were no longer differentially potent as discriminative stimuli in females and males, suggesting that factors that change over time (e.g., additional training, age, hormonal status) may contribute to initial sex differences in discriminability of U69,593.